Bottom Line:
Substances measured in EBC include oxidative stress and inflammatory mediators, such as arachidonic acid derivatives, reactive oxygen/nitrogen species, reduced and oxidized glutathione, and inflammatory cytokines.Although EBC is viewed as a noninvasive method for sampling airway lining fluid (ALF), validation is necessary to confirm that EBC truly represents the ALF.Likewise, a dilution factor for the EBC is needed in order to compare across subjects and determine changes in the ALF.

ABSTRACTExhaled breath condensate (EBC) has been increasingly studied as a noninvasive research method for sampling the alveolar and airway space and is recognized as a promising source of biomarkers of lung diseases. Substances measured in EBC include oxidative stress and inflammatory mediators, such as arachidonic acid derivatives, reactive oxygen/nitrogen species, reduced and oxidized glutathione, and inflammatory cytokines. Although EBC has great potential as a source of biomarkers in many lung diseases, the low concentrations of compounds within the EBC present challenges in sample collection and analysis. Although EBC is viewed as a noninvasive method for sampling airway lining fluid (ALF), validation is necessary to confirm that EBC truly represents the ALF. Likewise, a dilution factor for the EBC is needed in order to compare across subjects and determine changes in the ALF. The aims of this paper are to address the characteristics of EBC; strategies to standardize EBC sample collection and review available analytical techniques for EBC analysis.

fig3: Reactive oxygen and nitrogen species and redox relevant molecules in EBC. Exhaled nitric oxide (NO) is derived from L-arginine by enzyme nitric oxide synthase (NOS). NO can combine with superoxide (•O2−) to form peroxynitrite (ONOO−). ONOO− induces nitrosation of tyrosine (Tyr) residues and forms 3-nitrotyrosine (3-NT). NO can also react with thiols to form S-nitroso thiols (RS-NO). The end-products of NO are nitrite (NO2−) or nitrate (NO3−). •O2− is one of major reactive oxygen species generated from NADPH oxidase (NOX) or mitochondrial electron transfer chain. •O2− is converted to hydrogen peroxide (H2O2) by superoxide dismutases (SOD). H2O2 can be converted to the highly reactive hydroxyl radical (•OH), which is catalyzed by Fe2+ (Fenton reaction). H2O2 can be removed by thiol-specific antioxidant enzymes to form water.

Mentions:
Investigations of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are among the interests of EBC biomarkers in many lung disease studies. Multiple RNS formation starts with nitric oxide (NO). NO is a volatile component of the EBC [3, 13, 37, 38] and is synthesized from the amino acid L-arginine by nitric oxide synthase (NOS) (Figure 3). Different cell types within the respiratory tract have been identified to contain NOS, including airway and alveolar epithelial cells, macrophages, neutrophils, eosinophils, mast cells, and vascular endothelial and smooth muscle cells. Superoxide anion (•O2−) is a ROS that reacts quickly with NO, to form highly reactive peroxynitrite (ONOO−). ONOO− can cause the nitrosation of either tyrosine or tyrosine residues in proteins to form 3-nitrotyrosine (3-NT). Nitrotyrosine can be measured by enzyme immune assays or HPLC and MS [39–41]. NO can also react with thiols, such as cysteine, glutathione, or protein thiol residues to produce S-nitrosothiols (RS-NO) which can be measured by the colorimetric assay [42]. The end-products of NO metabolism are nitrite (NO2−) and nitrate (NO3−). In EBC, nitrite and nitrate can be measured by colorimetric, fluorometric, and chemiluminescent assays, or by ion, gas, and liquid chromatography [43, 44].

fig3: Reactive oxygen and nitrogen species and redox relevant molecules in EBC. Exhaled nitric oxide (NO) is derived from L-arginine by enzyme nitric oxide synthase (NOS). NO can combine with superoxide (•O2−) to form peroxynitrite (ONOO−). ONOO− induces nitrosation of tyrosine (Tyr) residues and forms 3-nitrotyrosine (3-NT). NO can also react with thiols to form S-nitroso thiols (RS-NO). The end-products of NO are nitrite (NO2−) or nitrate (NO3−). •O2− is one of major reactive oxygen species generated from NADPH oxidase (NOX) or mitochondrial electron transfer chain. •O2− is converted to hydrogen peroxide (H2O2) by superoxide dismutases (SOD). H2O2 can be converted to the highly reactive hydroxyl radical (•OH), which is catalyzed by Fe2+ (Fenton reaction). H2O2 can be removed by thiol-specific antioxidant enzymes to form water.

Mentions:
Investigations of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are among the interests of EBC biomarkers in many lung disease studies. Multiple RNS formation starts with nitric oxide (NO). NO is a volatile component of the EBC [3, 13, 37, 38] and is synthesized from the amino acid L-arginine by nitric oxide synthase (NOS) (Figure 3). Different cell types within the respiratory tract have been identified to contain NOS, including airway and alveolar epithelial cells, macrophages, neutrophils, eosinophils, mast cells, and vascular endothelial and smooth muscle cells. Superoxide anion (•O2−) is a ROS that reacts quickly with NO, to form highly reactive peroxynitrite (ONOO−). ONOO− can cause the nitrosation of either tyrosine or tyrosine residues in proteins to form 3-nitrotyrosine (3-NT). Nitrotyrosine can be measured by enzyme immune assays or HPLC and MS [39–41]. NO can also react with thiols, such as cysteine, glutathione, or protein thiol residues to produce S-nitrosothiols (RS-NO) which can be measured by the colorimetric assay [42]. The end-products of NO metabolism are nitrite (NO2−) and nitrate (NO3−). In EBC, nitrite and nitrate can be measured by colorimetric, fluorometric, and chemiluminescent assays, or by ion, gas, and liquid chromatography [43, 44].

Bottom Line:
Substances measured in EBC include oxidative stress and inflammatory mediators, such as arachidonic acid derivatives, reactive oxygen/nitrogen species, reduced and oxidized glutathione, and inflammatory cytokines.Although EBC is viewed as a noninvasive method for sampling airway lining fluid (ALF), validation is necessary to confirm that EBC truly represents the ALF.Likewise, a dilution factor for the EBC is needed in order to compare across subjects and determine changes in the ALF.

ABSTRACTExhaled breath condensate (EBC) has been increasingly studied as a noninvasive research method for sampling the alveolar and airway space and is recognized as a promising source of biomarkers of lung diseases. Substances measured in EBC include oxidative stress and inflammatory mediators, such as arachidonic acid derivatives, reactive oxygen/nitrogen species, reduced and oxidized glutathione, and inflammatory cytokines. Although EBC has great potential as a source of biomarkers in many lung diseases, the low concentrations of compounds within the EBC present challenges in sample collection and analysis. Although EBC is viewed as a noninvasive method for sampling airway lining fluid (ALF), validation is necessary to confirm that EBC truly represents the ALF. Likewise, a dilution factor for the EBC is needed in order to compare across subjects and determine changes in the ALF. The aims of this paper are to address the characteristics of EBC; strategies to standardize EBC sample collection and review available analytical techniques for EBC analysis.